Overrepresentation of CC proteins in the interactomes of Ure2, apCPEB, and Htt
The ubiquitin ligase CHIP, the chaperone Hsp104, the polyQ tract-binding protein-1 (PQBP-1), and the Htt-interacting protein (HIP-1) interact with Q/N-rich or polyQ proteins modulating their aggregation (Orr and Zoghbi, 2007
; Wickner et al., 2007
). In search of shared features among them, we discovered the common occurrence of CC domains, as previously found in three of them (e.g. Niu and Ybe, 2008
), and as predicted with high probability for PQBP-1 (). We extended our analysis to other interactors of the Q/N-rich prion Ure2 and the polyQ protein Htt listed in the BioGrid database, and found that 54% and 63% of Ure2 and Htt interactors, respectively, have or are predicted to have CC domains (). The apCPEB interactome is not well known, but two recently identified homologous or heterologous interactors, Hsp104 and Aplysia
CHIP are CC proteins (Si et al., 2003
; F.F. and E.R.K., unpublished). Taking into account interactors of CPEBs listed in the IntAct database, 84% of the putative apCPEB interactors are CC proteins (). Eukaryotic proteomes are estimated to contain only 6-8% CC proteins (Odgren et al., 1996
). Thus, our findings indicate that CC proteins are overrepresented among the interactors of these Q/N-rich and polyQ proteins.
Overrepresentation of CCs in Q/N-rich and polyQ proteins and in their interactomes
Q/N-rich and polyQ proteins contain heptad repeats typical of CC domains
Our interactome findings prompted us to search for CCs in the Q/N-rich prions and polyQ proteins themselves, as potential mediators of interaction. Using Coils (Lupas et al., 1991
) and Paircoil2 (McDonnell et al., 2006
), two algorithms that detect CC heptad repeats in primary sequences, we analyzed six yeast Q/N-rich prions (Ure2, Sup35, Rnq1, Swi1, Cyc8, Mot3), the Aplysia
Q/N-rich prion CPEB, and nine human proteins undergoing polyQ expansion (Htt, ataxin-1, -2, -3, -7, androgen receptor (AR), atrophin-1, calcium channel subunit α-1A (CACNA-1A), and TATA-box binding protein (TBP; Orr and Zoghbi, 2007
; Patel et al., 2009
; Alberti et al., 2009
). We found that all of these proteins contain heptad repeats and are predicted with high probability (0.8-1) to contain CC domains that flank/overlap with Q/N-rich regions and polyQ stretches (, S1
Heptad repeats are within prion domains (PrDs) of the yeast and Aplysia
proteins. In Ure2 and apCPEB, the CC region overlaps entirely with the regions defined as prion domains (, S1C
; Ure2 residues 1-95, Bousset et al., 2002
; apCPEB residues 1-160, Si et al., 2003a
). Swi1, Rnq-1, and Cyc8 display numerous regions of high CC propensity within their Q/N-rich domains (Fig. S1, S2
). Sup35 shows CC propensity within the M section of its NM prion domain (residues 124-253; Krammer et al., 2008; Fig. S1, S2
), which is crucial for prion propagation and Sup35 interactions with Hsp104 (Liu et al., 2002
). Moreover, 80% of the 19 candidate yeast prions identified by Alberti et al. (2009)
are predicted to have CC propensity (0.8-1, Paircoil2; and S2
All nine polyQ proteins show CC propensity in regions that contain polyQ stretches (, S1
). For some of them, the CC propensity of the wild-type (WT) polyQ regions is not high, but increases strongly upon polyQ expansion (Fig. S1B
). For comparison, we analyzed prions and amyloids devoid of Q/N-rich or polyQ stretches, such as the mammalian prion protein (PrP), β-amyloid(1-42), tau and α-synuclein (, S1
). Except for α-synuclein, already known to form CCs (Bussell and Eliezer, 2003
), these proteins do not show high CC propensity, suggesting that CC domains might represent a defining feature of the subgroup of Q/N-rich prions and polyQ amyloids. and S1A
show predictions for proteins with known CC crystal structures, including short CCs (c-Fos), long CCs (tropomyosin), and Q/N-enriched (>15%) CCs from the SIV protein gp41 and the Mycoplasma
protein MPN010 (Yang et al., 1999
; Shin et al., 2006
General features of predicted Q/N-rich coiled-coils
We compared the features of predicted Q/N-rich and poly-Q CCs with known CCs (). Coiled-coil heptads are repeats of seven residues (a-b-c-d-e-f-g
), in which hydrophobic residues (mostly L, I, V, M or F) often occupy positions a/d
, creating a hydrophobic layer between coiling helices (). Polar/charged residues are instead in solvent-exposed positions, and charged residues in e/g
can form CC-stabilizing salt bridges. Repeats of hydrophobic residues with heptad spacing are a hallmark of CCs, and are often discontinuous (Parry et al., 2008
, , S2G
). - Moreover, as seen in helical nets (e.g. gp41; ), hydrophobic residues in positions a/d
may be interspersed with certain non-hydrophobic ones such as Qs, also considered ambivalent hydrophobes (Sodek et al., 1972
). Q and N in position a/d
may specify the oligomeric state of CCs (Gonzalez et al., 1996
Heptad repeats in Q/N-rich and polyQ proteins
The putative Q/N-rich and polyQ CCs (, S2G
) contain a/d
clusters of hydrophobes alternating mostly with Q/Ns, with few charged residues in e/g
. Thus, predicted Q/N-rich and polyQ CCs have features comparable with known CCs, and suggest that they may be unstable and conformationally flexible (Li et al., 2003
; Kwok and Hodges, 2004
). A recurrent feature of these domains is the quite neat segregation of proline/glycine (P/G) residues outside of the CC-prone regions (Fig. S1C, S2D-E
We studied further the CC domains of Ure2, apCPEB, and human Htt, the determinant of Huntington’s disease upon polyQ expansion. We found that the N-terminal portions of Ure2, apCPEB and polyQ-expanded Htt exon-1 (Htt-72Q), are identified as CCs by Paircoil2 and contain 10, 11, and 12 heptad repeats, respectively (Fig. S3A-B
). Some heptads display a conventional amino acid composition, while others are quite atypical in this respect, being mostly formed by Q/N residues, either associated or not with hydrophobic residues in a/d
. Orthologs of these proteins are also predicted to form CCs, suggesting that these structures are evolutionarily conserved because of their functional relevance (Fig. S3C-D
Design of CC mutants
To study the role of CCs in the aggregation and activity of Ure2, apCPEB and Htt exon-1, we used structure-guided mutagenesis to modulate the stability of putative CC domains. We designed several mutants () and estimated CC disruption/enhancement for each using Paircoil2 (). In coiled-coil–defective (cc-) mutants, we substituted residues in a/d
with single or tandem prolines (), a commonly used strategy for CC disruption, impairing CC formation partially or completely (Chang et al., 1999
; ). Given the neat separation of conventional and Q heptads in Htt-72Q, we used this protein as a suitable model to modulate CC propensity also in the opposite direction. We generated CC-promoting mutants (cc+) predicted to i) induce aggregation of the soluble Htt-25Q, ii) enhance Htt-72Q aggregation, and iii) rescue aggregation of cc- mutants. Thus, we replaced 4-6 Qs with hydrophobic residues in the a-d
frame of heptads #1-2, thereby generating mutants Htt-25Q/cc+, Htt-72Q/cc+, and Htt-72Q/cc+R ().
Structure-guided mutagenesis of Ure,2 apCPEB, and Htt-72Q N-terminal domains
Since prolines in the cc- mutants may also affect β-sheet formation, we generated control mutants predicted to maintain propensity for CC (cc0), but in which the possible β-sheet formation would be impaired by replacing multiple Qs with β-sheet-breaking, charged or polar residues (Colloc’h and Cohen, 1991
). We used glutamate (E), lysine (K), and asparagine (N), up to four each, in various combinations (). To test the efficacy of these mutations in preventing β-sheet formation, we generated Aβ(1-42) mutants containing E, K, and N substitutions (β- mutants) with similar spacing as in the cc0 mutants (). These mutations disrupted the β-strand propensity of Aβ(1-42) (Fig. S3E, S5B-C
). As a further control for the proline mutagenesis, we generated the Htt-72Q/cc-/W mutant () in which four tryptophans (Ws) replaced the L-L-F-L pattern in a/d
. Tryptophan in a/d
is mildly CC destabilizing (Fig. S5E-F
; Kwok and Hodges, 2004
), but it has greater β-sheet propensity than leucine (L) (Pawar et al., 2007). Thus, this mutant would be expected to have preserved β-sheet-mediated aggregation but reduced CC-mediated aggregation.
Circular dichroism analysis of structure in Q/N-rich and polyQ peptides
To test the prediction that Q/N-rich and polyQ stretches can be part of α-helical CC structures, and to assess effects of the CC mutations, we used circular dichroism (CD) to analyze the secondary structure of three sets of peptides (). CD is used extensively to study the folding and stability of CCs. Distinctive signatures permit differentiating between single and coiled helices, based on the ratio between 222 and 208 nm ellipticities (>1 for CCs), its inversion induced by trifluoroethanol (TFE), and the thermal stability of the folding (see Suppl. Experimental Procedures
Secondary structure and oligomeric state of Q/N-rich and polyQ peptides
The first set of peptides is based on a four-heptad CC model (Hicks et al, 2002) into which we inserted two central heptads of Qs (peptide ccQQ), or Qs with either CC-stabilizing (I-L-I-L) or -destabilizing (P-P-P-P) residues in a/d
(ccQL and ccQP, respectively). In benign buffer, ccQQ and ccQL displayed α-helical CD spectra with minima at 208 and 222 nm, whereas ccQP showed a minimum at ~200 nm as for random coils (). Both ccQQ and ccQL had θ222
ratios >1, indicating CC formation. In 50% TFE, which favors CC dissociation, the θ222
ratio of ccQQ and ccQL went below 1 (, S4A
), as typically observed for CCs. TFE also induced helical folding of ccQP, likely by stabilizing its proline-flanking parts (Fig.S4A
). Expected CC stabilization in ccQL vs
ccQQ was realized; ccQL remained folded up to 75°C, whereas ccQQ lost most of its structure already at 25 °C (). We conclude that polyQ stretches can be part of CC structures, and that Q-rich CCs gain stabilization by hydrophobic residues with heptad spacing.
We similarly analyzed Ure2 and Htt peptides. Ure2(70-98), comprising heptads #7-10 (Fig. S3A
), is only partially helical at 4°C, and becomes essentially unstructured above 25°C (, S4B
). Difference spectra (37°C vs. 4°C) revealed more clearly that the structured component is α-helical and coiled, as indicated by a θ222
ratio ~1 (). This conclusion is also supported by the inversion of the ratio in 50% TFE, which also strongly enhanced α-helical folding (Fig. S4B
). Htt-17Q (1-40), designed by reference to the only form of Htt that has been crystallized (Kim et al., 2009
), generated CD spectra similar to Ure2(70-98), being only partially helical at 4°C, consistent with the crystal structure (Kim et al., 2009
; Fig. S4C-D
). Interestingly, unlike the other peptides, the CD spectra of Htt-17Q changed with time, showing a reduction in ellipticity at 208 nm and a progressively increasing θ222
ratio. After some hours at 4°C, the CD spectra showed a helical profile with θ222
>1, indicative of the CC formation, and moderate thermal stability (). Subsequently, θ208
decreased further and the 222 nm minimum was red-shifted towards 225 nm (Fig. S4C-D
); similar transitions have been observed in CD spectra of peptides forming CC fibers (e.g. Potekhin et al., 2001
; Frost et al., 2005
Finally, to assess the structural consequences of the CC-targeting mutations, we studied peptides derived from cc+ and cc- mutants. Htt25Q/cc+(1-31) displayed frank CC features (θ222/θ208 ratio > 1) and notable thermal stability (). Conversely, Htt72Q/cc-/#2(1-31), comprising the first four heptads of Htt-72Q with prolines in a/d, displayed a random coil conformation ().
Quantitative fittings for CD spectra of peptides with θ222
ratio > 1 (ccQQ, ccQL and Htt-25Q/cc+(1-31), Supplemental Table S1
) confirm high helical content (40-75%) and show negligible levels of β-sheet structure (2-8%). Moreover, the thermal denaturation series () have isodichroic points at ~203 nm, typically found upon CC destabilization and indicative of an equilibrium between α-helices and random coils (Gazi et al., 2008
). These results are in excellent accordance with predictions for Ure2 and Htt ().
Cross-linking analysis of oligomerization by CC-forming polyQ and Q-rich peptides
CC assemblies range from dimers to polymers (Parry et al., 2008
). To define the oligomeric state of the peptides characterized by CD, we performed glutaraldehyde cross-linking experiments, which revealed dimeric and higher-order species (). At 37°C, ccQL and Htt-25Q/cc+(1-31) are mostly in higher-order multimers; ccQQ forms supradimeric species as well, but less so than ccQL, consistent with its lower stability ().
Non-cross-linked Htt-17Q(1-40) ran as dimers, although higher order forms were also present (, arrow). Cross-linked Htt-17Q(1-40) generated an opalescent solution with visible aggregates indicative of large peptide assemblies, which were not able even to enter the gel (, white asterisk). These and CD results demonstrate the marked polymerization tendency of Htt-17Q(1-40).
Ure2(70-98), which is marginally folded at 4°C, did not display significant formation of supradimeric species at 37°C (not shown), in accord with CD. The proline-containing peptides ccQP and Htt-72Q/cc-/#2(1-31) were mostly monomeric, displaying only a very modest degree of dimerization ().
These findings show that α-helical CC-prone polyQ and Q/N-rich peptides form higher-order oligomers in vitro, and indicate the possibility that CCs may trigger protein aggregation in vivo.
CC destabilization hampers in vivo aggregation of Ure2, of apCPEB, and Htt-72Q
To determine the relevance of CCs for in vivo aggregation, we compared the subcellular distribution of WT and cc- mutant Ure2, apCPEB and Htt-72Q overexpressed as GFP fusions.
WT and cc- Ure2 were expressed in a knock-out yeast strain (ure2Δ) to prevent any influence of endogenous Ure2. Ure2-GFP formed aggregates in numerous cells (25.7 ± 1.1%, n=36 50×50 μm microscopic fields of cultures from 3 colonies; ), whereas the cc- mutant had mostly diffuse subcellular distribution, forming aggregates in significantly fewer cells (p<0.01, t-test; ).
CC disruption impairs aggregation of Ure2, apCPEB, and Htt72Q
Aggregation of CC-enhancing and CC-neutral mutants
We next overexpressed WT or cc- mutant apCPEB in Aplysia
neurons (). ApCPEB formed aggregates in the soma, rapidly decreasing in number along the axon, and no diffuse fluorescence was detectable between them, or in distal neuritic branches (). Conversely, mutant cc-/#2 presented diffusely along the neurites (), down to distal branches where no aggregate was detectable. Mutant cc-/#1, predicted to disrupt CC only partially (), had an intermediate phenotype (data not shown). The cc-/#2 mutant still formed aggregates proximally to the cell body, similar to what observed on overexpressing truncated apCPEB devoid of its Q/N-rich domain (Si et al., 2010
). These results are similar to what is found for a non-Q/N-rich RNA-binding protein (PABPN-1), whose aggregation relies both on an N-terminal CC and a C-terminal RNA-binding domain (Tavanez et al., 2005
Finally, we compared the distribution of Htt-72Q and its cc- mutants in HEK293 cells 72h after transfection (). Aggregation was significantly impaired for mutant cc-/#1, and almost completely abolished for cc-/#2. ANOVA () showed an overall effect of the CC mutations on aggregation (F(12,337)
= 103.11, p<0.001), with significant differences between Htt-72Q and both its cc- mutants (p<0.01, Newman-Keuls test). These phenomena were not cell-type-specific (, S5A
). In addition, we tested the role of CCs in the heterotypic interactions of Q/N-rich and polyQ proteins. We found that CC fragments of interactors are recruited into aggregates (Fig. S6A-B
) and that CC destabilization impairs the Htt-72Q/CHIP interaction (Fig. S6C
). These findings demonstrate a close correlation between CC propensity of Ure2, apCPEB, and Htt-72Q and their aggregation properties.
Enhancing CC propensity in Htt induces or increases aggregation
We next analyzed the aggregation of CC-enhancing (cc+) mutants. Htt-25Q does not aggregate upon overexpression, but the Htt-25Q/cc+ mutant formed multiple aggregates per cell in a proportion of cells not different from the Htt-72Q group (), showing that a non-aggregating form of Htt can be induced to aggregate by increasing its CC propensity, independently of polyQ expansion. The possibility that this aggregation was a consequence of β-sheet formation induced by the additional hydrophobic residues is ruled out by the CD results (). Furthermore, we found that the addition of the same L-L-F-L pattern with heptad spacing amidst the polyQ stretch of Htt-72Q (Htt-72Q/cc+) increased aggregation (p<0.01), and that a similar pattern plus an additional F-L pair (Htt-72Q/cc+R) was able to substantially rescue the aggregation of cc-/#1 (p<0.001; ).
These experiments show that increasing the CC propensity of Q stretches can induce or enhance aggregation in vivo, and they support the notion that Q stretches may be part of CCs, especially when stabilized by flanking or overlapping heptad repeats of hydrophobic residues.
β-Sheet breaking residues in the polyQ stretch of Htt-72Q do not abolish aggregation
To rule out that the effect of proline substitutions in cc- mutants may be due to β-sheet disruption rather than CC destabilization, we studied the effect of mutations disfavoring β-sheet but not CC formation (cc0 mutants), and compared the effect of the same mutations on the aggregation of Aβ(1-42), a known β-sheet-forming amyloid (β-mutants).
All cc0 mutants formed aggregates after 72 hours of overexpression () in ~50-60% of the cells (i.e. 70-85% of the Htt-72Q aggregation rate), a significantly higher proportion than for cc- mutants (p<0.01, Newman-Keuls test). Interestingly, the microstructure of cc0 aggregates became more elaborate as the number of substitutions increased, showing spiny protrusions, star-like figures, and eventually tangled fibers ().
On the other hand, while GFP-Aβ(1-42) aggregated in many of the overexpressing cells (17.71 ± 2.51%, n=20 fields; 391 cells), the aggregation of β- mutants #1 and #2 was completely abolished (p<0.01; , S5D
), thus demonstrating the efficacy of the cc0 mutations in disrupting β-sheet-based aggregation. Furthermore, Htt-72Q/cc-/W formed smaller aggregates than Htt-72Q (, S5E-G
), as expected for CC-driven aggregation since L→W substitution is mildly CC destabilizing but β-sheet enhancing.
These findings further support the notion that the aggregation of Q-rich proteins relates essentially to their CC propensity and that conversion to β-sheets may not be crucial for triggering aggregation.
Modulation of CC propensity alters detergent-insolubility of Ure2, apCPEB, and Htt-72Q
Aggregated amyloids display detergent insolubility, which can be assayed by ultracentrifugation of cell lysates to separate detergent-insoluble aggregates from soluble forms. We collected lysates of yeast and HEK293 cells overexpressing Ure2 and Htt variants for 72 hours. Given the lack of mass transfection systems for Aplysia cells, we expressed apCPEB variants in HEK293 cells, which were lysed 12-18 h after transfection, given the faster aggregation kinetics of this protein in these cells.
WT Ure2, apCPEB, and Htt-72Q were detectable in both soluble and insoluble fractions, (). However, all cc- mutants displayed a remarkable reduction of the detergent-insoluble fraction (). The data for Htt mutants were normalized to Htt-72Q (65.6 ± 2.4% in the pellet fraction, n=13), and revealed an overall significant effect of the mutations on solubility (F(10,55)=33.95, p<0.001; one-way ANOVA ). The insoluble fraction of the cc-mutants was strongly reduced with respect to Htt-72Q (p<0.001, Newman-Keuls test). Conversely, the proportion of the cc+ mutant in the pellet was significantly increased as compared with Htt-72Q (p<0.03), and, the cc+R mutant showed a rescued insolubility with respect to cc-/#1 (p<0.001). Finally, cc0 mutants were found in the pellet fraction in a proportion >80% of that of Htt-72Q, significantly more than cc- mutants (p<0.001).
CC propensity regulates insolubility and protein activity/toxicity
Thus, the detergent insolubility correlated with CC propensity, closely paralleling the aggregation phenotypes. These findings are also consistent with other observations of detergent-insoluble structures from CC proteins (e.g. Yang et al., 2002
CC disruption impairs [URE3] prion formation and abolishes Htt-72Q-induced cytotoxicity
We next sought to determine whether CC disruption also interferes with Ure2 function. We used ureidosuccinate (USA) uptake to monitor the presence of [URE3] in Ure2Δ cells overexpressing WT or mutant Ure2, grown under plasmid selection on either uracil or USA substrate (). Both WT and mutant transformants grew equally well on the non-selective uracil-containing substrate, whereas cc- transformants grew significantly less on USA. The cc- transformants formed about half of the colonies formed by WT Ure2 transformants, as determined at dilutions from 10-3 to 10-5 (51.2 ± 2.5%, n=18 vs 100 ± 4.6%, n=30, p<0.001, t-test; ). These results indicate that the N-terminal CC structure of Ure2 is important not only for aggregation but also for prion induction.
The cytotoxicity of polyQ-expanded proteins can be assayed in vitro; thus, we tested the impact of CC mutations on the activity of Htt-72Q using a colorimetric assay to detect the cellular reduction of MTT to formazan. We observed significant cytotoxicity in HEK293 cells after 72h of Htt-72Q overexpression; formazan production was significantly less after Htt-72Q transfection than in mock-transfected control cultures (i.e. 80.4 ± 1.8% in n=45 culture wells versus 100 ± 1.5% for n=46 controls, p<0.001 t-test). Then, we compared the Htt-72Q cytotoxicity with that of its CC mutants, normalizing the toxicity of each mutant to that of Htt-72Q (). ANOVA revealed a significant overall effect of the mutations on Htt-72Q toxicity (F(11,402)=13.10, p<0.001). The cc- mutants were the only two Htt-72Q mutants devoid of toxicity, whereas all the other mutants retained substantial toxicity. The toxicity of cc-/#1 and /#2 was not different from the control group (p>0.42). The cc+R mutant was significantly toxic with respect to control (p<0.04), as well as the cc+ mutant (p<0.001). The cc0 mutants all displayed significant toxicity, ranging between 42 and 63% of the Htt-72Q toxicity (p<0.02).
These results show a strong correlation between the CC propensity of Htt-72Q variants and their toxicity, similar to that we observed for their aggregation and insolubility. The essential role of the non-polyQ heptads in mediating Htt-72Q dysfunctional activity is evident in the fact that their disruption alone (cc-/#1) was sufficient to abolish toxicity.